1. Field
This invention is concerned generally with the immobilization of catalytically active enzymes onto essentially water insoluble carrier materials. Specifically, the invention is concerned with the adsorption of glucose isomerase onto the internal surfaces of a high surface area, porous inorganic support consisting of mixed metal oxides of MgO and Al.sub.2 O.sub.3.
Because of the catalytic specificity of enzymes, considerable attention has been directed toward finding methods of using them in both laboratory and industrial applications. Enzymes are commonly water-soluble, and, for that reason, many enzymes are uneconomical to use in large scale batch-type operations since the enzymes can generally be used only one time in the absence of rather costly enzyme recovery and purification steps. In recent years, however, techniques have been devised to fix active enzymes on essentially water-insoluble support materials that can be readily removed from a reaction, thus permitting re-use of the insolubilized or immobilized enzyme. This disclosure describes a novel immobilized glucose isomerase composite demonstrating a high degree of enzymatic activity per unit weight of composite.
2. Prior Art
Glucose isomerase is an enzyme which catalyzes the isomerization of the sugar glucose to the sugar fructose, sometimes referred to as levulose. The desirability of converting glucose to fructose is well recognized for various reasons. For example, although fructose has the same caloric value as glucose, it is a sweeter sugar. Thus, with fructose, a fixed sweetness can be achieved at a relatively lower caloric intake. Further, the sugar glucose is relatively abundant from a variety of sources and, hence, available as a raw material source for fructose production.
It has been well known that glucose can be isomerized to fructose by both alkaline and enzymatic methods. The alkaline isomerization of glucose solution requires subjecting a glucose-containing solution to an alkaline environment in which isomerization to fructose can occur. Unfortunately, the alkaline isomerization of glucose to fructose has been unsatisfactory, generally because of the tendency of non-selective alkaline catalysts to produce undesirable by-products which adversely affect the product taste and which are difficult to remove. Among the known undesirable byproducts of alkaline isomerization are various color bodies and acidic products which require added processing steps for removal. Some of the disadvantages associated with alkaline isomerization have been overcome by the relatively recent discovery that finely divided alumina may be used in an alkaline environment to isomerize glucose. As disclosed in U.S. Pat. No. 3,431,253, by using alkaline alumina (&gt;pH 7) having a large surface area, it has been found possible to avoid formation of objectionable by-products. A further disclosed advantage is that by using solid alumina particles, the alumina can easily be removed from a reaction medium and reused. Unfortunately, however, the use of finely-divided alumina requires a relatively long residence time for the glucose solution, thus tending to preclude more economical continuous or flow-through reactions. Further, because of a suspected equilibrium which exists between glucose and fructose, the optimum conversion of glucose to fructose is limited under the batch-type reaction conditions disclosed in the above patent.
Because of the problems associated with alkaline isomarization, increasing attention is being directed toward enzymatic isomerization methods using glucose isomerase. As used herein, glucose isomerase refers to that enzyme or enzyme system which catalyzes the isomerization of glucose to fructose, regardless of enzyme source. The enzyme itself can be derived from a variety of organisms (e.g., U.S. Pat. No. 3,813,318) and numerous methods are known for extracting and purifying glucose isomerase. The use of soluble glucose isomerase preparations for large scale commercial fructose manufacture is generally limited due to enzyme cost for one-time use and/or costs associated with recovery or inactivation of the spent soluble enzyme. For those reasons, recent attention has been directed toward finding methods of immobilizing enzymes on high surface area, essentially water-insoluble carrier materials, both organic and inorganic.
There are a number of disadvantages associated with the use of organic carriers as enzyme support materials. For example, many organics are subject to microbrial attack, especially during long term use. Further, some of the organics tend to swell in an aqueous environment, thus posing pressure problems in continuous use column operations. Further yet, many organics lack a very high surface area needed to assure maximum enzyme loading and, because of their organic nature, many such carriers are difficult to sterilize by conventional methods. Many of the above disadvantages have been overcome by recent discoveries showing that certain inorganic materials can be used as enzyme support materials.
Methods of adsorbing various enzymes to a number of siliceous materials are disclosed in U.S. Pat. No. 3,556,945. Methods of chemically coupling enzymes to a wide variety of inorganics through an intermediate silane coupling agent are disclosed in U.S. Pat. No. 3,519,538. More recently in patent application Ser. No. 332,807, filed Feb. 16, 1973, now U.S. Pat. No. 3,850,751, entitled "Enzymes Immobilized on Porous Inorganic Support Materials," filed in the name of R. A. Messing and assigned to the present assignee, it was disclosed that very efficient immobilized enzyme composites could be prepared by bonding the enzymes to the internal surface of porous ceramic materials having an average pore diameter of less than 1000 A, preferably less than about 500 A or between about 100 A and 500 A. By choosing an average pore diameter at least as large as the size of the enzyme but less than about 1000 A, it was disclosed that a high surface area for high enzyme loading is provided and that the internally bonded enzymes tended to be protected from detachment, especially in turbulent reaction environments.
In U.S. Patent Application S. N. 332,739, filed Feb. 16, 1973 now U.S. Pat. No. 3,868,304 in the name of R. A. Messing, entitled "Method of Making Fructose with Immobilized Glucose Isomerase," and assigned to the present assignee, there is disclosed a method of isomerizing glucose to fructose using a composite consisting of glucose isomerase adsorbed to porous alumina bodies having an average pore size ranging from about 100 A to 1000 A. As disclosed in the above-cited patent application, it is known that various metal ions are needed in the enzymatic isomerization of glucose to fructose. See, for example, an article by Y. Takasaki et al., entitled, "Studies on Sugar-Isomerizing Enzyme, Purification, Crystallization and Some Properties of Glucose Isomerase from Streptomyces sp.," in Agr, Biol. Chem., Vol. 33, No. 11, p. 1527-34 (1969). In that article, the effects of various metal ions such as Mg, Co, Fe, Mn, Ni, Ba, Ca, Zn, and Cu, were examined with the conclusion that glucose isomerase from a cited Streptomyces sp. strain requires the presence of both cobalt and magnesium ions for its activity. Typically, the presence of those and other ions is assured by adding them to the glucose feed prior to contact with an immobilized enzyme system, as shown by the examples of Ser. No. 332,739. The addition of various metal ions to the glucose feed material requires added processing steps in the production of fructose with immobilized glucose isomerase, especially in a continuous flow-through reactor (e.g. plug-flow column). Further, when such ions are added to the feed solution, they become a part of the final product. This is especially undesirable in the case of ions such as those of cobalt. Because of the undesirability of having such ions in the final product, especially food products, and because of added costs associated with the removal of such ions, there has been a recognized need for providing an immobilized glucose isomerase system which assures the presence of needed metal ions and yet avoids the problems associated with past systems.
Quite surprisingly, it has been found that apparently a portion of the metal ion requirements of an immobilized glucose isomerase system and the critical pH parameters of such a system can be simultaneously met with a porous inorganic carrier material having a very critical metal oxide composition range, described in detail below.